The objectives of this project were the characterization of two recently cloned genes and the expression of a previously cloned gene under the control of a heat shock promoter (hsp) from Coxiella burnetii. A. Gene cloning. Genomic DNA potentially encoding aspartate transcarbamoylase (ATCase, pyrB) was cloned into the plasmid pEMBL8+. A 7 kb fragment was selected for its ability to complement a pyrB-E. coli auxotroph. The putative C. burnetii pyrB gene was shown to express an active ATCase using cell-free extracts of the recombinant E. coli. DNA sequence analysis of a 2.2 kb subclone revealed a product of approximate size and composition in common with the pyrB gene of E. coli. B. Surface antigen. The product of a 2.5 kb fragment from the same EcoR1 genomic library was selected by monoclonal antibodies raised against a 29.5 kd immunodominant surface protein. DNA sequence analysis revealed an open reading frame encoding a 30 kd lipoprotein. The amino acid composition of the purified 29.5 kd surface protein conflicts with the deduced amino acid composition of the cloned lipoprotein gene product detected by monoclonal antibodies. C. HSP. A recently cloned operon, htpAB, under the control of an hsp from C. burnetii expressed 62 and 14 kd gene products at 42 degrees C in the recombinant E. coli. A similar study in C. burnetii indicated that both of these and at least 5 more genes were maximally expressed at 42 degrees C. In addition a cold-shock gene product was only expressed at 23 degrees C. D. Significance. The C. burnetii pyrB gene resembles structurally the gene of E. coli, but appears to be regulated differently. An immunodominant 29.5 kd surface protein may share a common epitope with other similarly sized lipoproteins. The induction of gene expression during stress responses under the control of hsp may contribute to the survival of C. burnetii in the phagolysosome.